Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.      

The structure, catalytic mechanism and regulation of adenylyl cyclase

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Insights into wolf presence in human‐dominated landscapes: the relative role of food availability,humans and landscape attributes

Aim Understanding which human or environmental factors interact to enable or to limit the occurrence and persistence of large carnivores in human‐dominated landscapes is an important issue for their effective conservation, especially under the current scenario of global change where most of their former habitat is being transformed by humans. Location NW Iberian Peninsula. Methods We combine data on the distribution of Iberian wolves (Canis lupus signatus) living in a human‐dominated landscape in NW Spain and variation and partitioning methods to investigate the relative importance of three groups of predictors: food availability, humans and landscape attributes – each group expected to have unequal effects on wolf reproduction and survival – and their interactions on the occurrence of this species. Results We found that the group of predictors related with landscape attributes (altitude, roughness and refuge) strongly determined wolf occurrence, followed by humans and food availability. Variance partitioning analysis revealed that the three most important components determining wolf occurrence were related with landscape attributes: (1) the joint effects of the three predictor groups, (2) the joint effect of humans and landscape attributes and (3) the pure effect of landscape attributes. Altitude had the main independent contribution to explain the probability of wolf occurrence. Main conclusions In human‐dominated landscapes, the occurrence of wolves is the result of a complex interaction among several environmental and human factors. Our results suggest that the characteristics of the landscape (spatial context) – factors associated with the security of wolves facilitating that animals go unnoticed by humans, wolf movements, dispersal events and short‐time colonization – become more important in human‐dominated landscapes and may have played a key role in the occurrence and persistence of this species throughout decades modulating the relationship between humans and wolf distribution.     

The importance of fine-scale breeding site selection patterns under a landscape-sharing approach for wolf conservation

For large carnivores persisting in human-dominated landscapes, conservation planning is often hindered by the large spatial requirements of these species, availability of protected areas, and human land uses. Protected areas are usually too small to support viable populations, and scattered across a human land-use matrix. Therefore, large carnivore conservation should be planned at large spatial scales under a land-sharing approach (allowing the coexistence between large carnivores and people in the same landscape), which means increasing the focus on the human-dominated matrix. Most of the critical factors determining large carnivore persistence (i.e., those related to food availability and vulnerability to humans) interact synergistically in space and time during the breeding season. Here, using as a case study a wolf population in NW Iberia, we studied fine-scale breeding site selection patterns (1 and 9 km²) in relation to human pressure, and the availability of food and refuge. The selection of wolf breeding sites in this human-dominated landscape was not determined by potential availability of prey biomass in the immediate vicinity (1 km²). However, wolves selected breeding sites with high availability of refuge (concealing vegetation), and low human accessibility and activity levels. Paved roads showed the highest proportion of independent contribution to explaining breeding site selection patterns (negative influence), being followed by refuge availability (positive influence) and the remoteness of breeding sites in relation to the surrounding spatial context (positive influence). Refuge availability, even at very small spatial scales taking into account the spatial requirements of wolves, may compensate for moderate levels of human activities in the vicinity of breeding sites. The strength of breeding selection patterns varied along a hierarchical process at different spatial scales. Under a landscape-sharing approach, integrating key processes observed in the human-dominated matrix, such as breeding site selection patterns, into landscape planning is of paramount importance for carnivore conservation. By temporally restricting human use on breeding sites and small portions of surrounding lands (~ 1 km²), and by maintaining several small refuge areas interspersed within the human-dominated matrix, we could favor wolf persistence without reducing land availability for other uses, improving the conditions for coexistence between wolves and humans.     

Resting in risky environments: the importance of cover for wolves to cope with exposure risk in human-dominated landscapes

Centuries of persecution have influenced the behaviour of large carnivores. For those populations persisting in human-dominated landscapes, complete spatial segregation from humans is not always possible, as they are in close contact with people even when they are resting. The selection of resting sites is expected to be critical for large carnivore persistence in human-dominated landscapes, where resting sites must offer protection to counteract exposure risk. Using wolves (Canis lupus) as a model species, we hypothesised that selection of resting sites by large carnivores in human-dominated landscapes will be not only influenced by human activities, but also strongly determined by cover providing concealment. We studied the fine-scale attributes of 546 wolf resting sites and confronted them to 571 random points in NW Iberia. Half of resting sites (50.8 %) were found in forests (mainly forest plantations, 73.1 %), 43.4 % in scrublands, and only 5.8 % in croplands. Compared to random points, wolves located their resting sites far away from paved and large unpaved roads and from settlements, whereas they significantly selected areas with high availability of horizontal (refuge) and canopy cover. The importance of refuge was remarkably high, with its independent contribution alone being more important than the contribution of all the variables related to human pressure (distances) pooled (51.1 vs 42.8 %, respectively). The strength of refuge selection allowed wolves even to rest relatively close to manmade structures, such as roads and settlements (sometimes less than 200 m). Maintaining high-quality refuge areas becomes an important element to favour the persistence of large carnivores in human-dominated landscapes as well as human-carnivore coexistence, which can easily be integrated in landscape planning.